There are dozens of inhibitors of epigenetic regulation in clinical trials. However, the effects of these inhibitors on global epigenetic silencing of chromatin have not been investigated. Because understanding the effects of inhibition of epigenetic regulators is clearly of critical clinical importance and because genome-wide analyses of the effects of inhibiting various mechanisms of epigenetic regulation are essentially non-existent, we will perform genome-wide epigenetic analyses before and after inhibition of histone methyltransferases (HMTases), DNA methyltransferases (DNMTases), and histone deacetylases (HDACs). We will investigate the effects of the HDAC, HMTase, and DNMTase inhibitors on global chromatin structure and determine if inhibition of one type of chromatin repression mechanism affects other mechanisms of repression. We will also determine if inhibition by a drug produces identical results as that obtained using cells nullizygous for the targeted enzyme. These questions will be explored in Specific Aim I, using mouse embryonic stem cells (mES cells) as our model system. The pluripotency of ES cells has engendered great excitement concerning their potential for cell replacement therapies for a variety of human diseases. Specifically, it has been proposed that human embryonic stem cells (hES) may provide a good source for functional hepatocytes for repopulation of diseased human livers. However, it has also been suggested that cancer evolves from a population of epigentically altered stem cells. To date, comprehensive comparisons of in vitro differentiated cells with their normal adult counterparts have not been performed. Because of the clinical importance of this issue, it is critical that the in vitro differentiated hepatocytes be monitored for [unreadable]epigenetic accuracy[unreadable] before transplanting into a patient. Therefore, in Specific Aim II, we propose to map the active and silenced regions of the genome in proliferating hES cells, hES cells that have been differentiated into hepatocytes, normal hepatocytes from adult human liver, and liver tumor tissue. By comparing the epigenetic profiles of these different cell populations, we will be able to test the hypothesis that in vitro differentiated hepatocytes are a good mimic of adult hepatocytes.